This invention relates to the use of oil bodies and their associated proteins as affinity matrices for the separation and purification of target molecules from samples.
Within the general field of biotechnology, the ability to effectively separate and purify molecules from complex sources, such as living cells, blood serum, or fermentation broth, is of critical importance. Applications in industry and research laboratories (where, for example, purified or partly purified proteins are used) are numerous and well documented in prior literature. See, for example, R. Meadon and G. Walsh in Biotechnological Advances 1994, 12: pp 635-646.
The majority of currently employed techniques for the separation of molecules capitalizes on the innate physical and chemical properties of the molecule of interest. Affinity-based purification technologies are unique in that they exploit the highly specific biological recognition between two molecular species which form an affinity pair. Binding of the two entities of the affinity pair occurs in almost all instances as a result of relatively weak chemical interactions, known as non-covalent bonds. Some art-recognized and commonly used affinity pairs include antibodies and their binding antigenic substances, nucleic acid binding proteins and nucleic acids, lipid binding proteins and lipids, lectins and carbohydrates, streptavidin/biotin complexes, protein A/immunoglobulin G complexes, and receptors and their binding molecules.
In general, affinity-based purification processes require that one member of the affinity pair is immobilized on a solid substrate or matrix that is insoluble in the fluid in which the other member of the pair resides. The molecular species of the affinity pair bound to the matrix is generally referred to as the ligand, while the liquid soluble member is generally referred to as the target member. However, it is important to note that these definitions do not impose any restrictions in a strict chemical sense. The vast majority of current ligand immobilization techniques rely on physical or chemical approaches. Physical ligand immobilization involves adsorption or entrapment of the ligand to a suitable support, while the chemical mode of immobilization is characterized by the formation of strong crosslinks or covalent attachments between the ligand and the matrix. It is a requirement that immobilization is accomplished in such a fashion that the capacity of the members of the affinity pair to recognize each other is not adversely affected by the immobilization procedure.
It is a disadvantage of the currently available physical and chemical techniques for immobilizing ligands that production processes are frequently time consuming and expensive. This is mainly due to the fact that immobilization techniques require the separate production of matrix material and ligands, which in a subsequent step must be coupled. An alternative mode of immobilizing proteins is described in U.S. Pat. No. 5,474,925 which documents a biological production system for the immobilization of enzymes in the fibre of cotton plants. This patent discloses what is believed to be the first biologically produced enzyme immobilization system and allows a one step production of matrix and ligand.
Subsequent to immobilization of the ligand on the matrix, a variety of affinity based purification techniques may be employed to accomplish selective binding between the affinity immobilized ligand and the target member. Affinity based purification techniques known in the prior art include perfusion affinity chromatography, affinity repulsion chromatography, hyperdiffusion affinity chromatography, affinity precipitation, membrane affinity partitioning, affinity cross-flow ultrafiltration and affinity precipitation. In the most widely used affinity based purification technique, affinity chromatography, a matrix containing a ligand is coated to, or packed on, the inside of a chromatographic column. A complex mixture containing the target member is then applied to the chromatographic column. Ideally, only the target molecules that specifically recognize the ligand bind in a non-covalent fashion to the chromatographic column, while all other molecular species present in the sample pass through the column.
In affinity partitioning, two solutions of substantially different densities are employed. The complex solution containing the target member is mixed with a solution of a different density containing the affinity ligand. Subsequent to mixing, the solutions are left to settle in order to permit the formation of two separate phases. Molecules tend to partition differentially between phases depending on their size, charge and specific interactions with the phase-forming solutions. Ligand-bound target protein selectively partitions to the phase containing the affinity ligand. For example, Coughlin and Baclaski in Biotechnology Progress, 1990 6: 307-309 reported the use of the biotin containing organic solution isooctane to transfer avidin from an aqueous solution to the isooctane solution. However, so far applications of affinity partitioning have been limited mainly due to the current lack of availability of suitable affinity matrix substances which can be employed in specific partitioning in two phase systems.
An important factor for the commercial development of biotechnology is the purification of bioproducts, which typically accounts for 50% or more of the total costs (Labrou, N. and Clonis, Y. D. in the Journal of Biotechnology 36: 95-119 (1994)). Many protein purification steps rely on column type separation procedures. In particular, large scale high-separation techniques such as column chromatography or batch-type based protein purification techniques are costly. In addition, crude material is less suitable for either column chromatography or batch separations, as contaminants may foul up sedimented resins and plug columns. Thus, affinity matrices are often only employed in a later stage of purification processes where substantial purity is critical, where the proteins are present in extremely dilute concentrations, or where high value proteins are required, for example in diagnostic and therapeutic proteins. These and other topics related to the use of affinity technology in biotechnological processes have been reviewed by Labrou, N. and Clonis, Y. D. in the Journal of Biotechnology 36: 95-119 (1994).
There is a need in the art to develop novel and economical methods for separating and purifying biological products from complex mixtures. The present inventors have found that subcellular oil storage structures, known as oil bodies, and their associated proteins are useful in this regard.
The present invention relates to a novel versatile biological system for the production of affinity matrices. The present inventors have found that oil bodies and their associated proteins can be used as affinity matrices for the separation of a wide variety of target molecules such as proteins, carbohydrates, lipids, organic molecules, nucleic acids, metals, cells and cell fractions from a sample.
In accordance with the invention, there is provided a method for the separation of a target molecule from a sample comprising: 1) contacting (i) oil bodies that can associate, either directly or indirectly, with the target molecule with (ii) a sample containing the target molecule; and 2) separating the oil bodies associated with the target molecule from the sample. The oil bodies and the sample containing the target molecule are brought into contact in a manner sufficient to allow the oil bodies to associate with the target. Preferably, oil bodies are mixed with the target. If desired, the target molecule may be further separated from the oil bodies.
In one aspect, the target molecule has affinity for, or binds directly to, the oil bodies or oil body protein. Examples of such targets include antibodies or other proteins that bind to oil bodies.
In another aspect, a ligand molecule may be used to associate the target molecule with the oil bodies.
In one embodiment, the ligand has natural affinity for the oil bodies or oil body protein. In a specific embodiment, the ligand is an antibody that binds the oil body protein. Such an antibody can be used to separate targets having affinity for the ligand antibody such as anti-IgG antibodies or protein A. A bivalent antibody may also be prepared having binding specificities for both the oil body protein and the target. The antibody against the oil body protein may also be fused to a second ligand having affinity for the target.
In another embodiment, the ligand is covalently attached to the oil bodies or oil body protein. In one embodiment, the ligand is a protein that is chemically conjugated or produced as a fusion protein with the oil body protein (as described in WO 96/21029). In the latter case, the fusion protein is targeted to and expressed on the oil bodies. In one example, the ligand fused to the oil body protein may be hirudin and can be used to purify thrombin. In another example, the ligand fused to the oil body protein may be metallothionein and can be used to separate cadmium from a sample. In a further example, the ligand fused to the oil body protein may be protein A and can be used to separate immunoglobulins. In yet another example, the ligand fused to the oil body protein may be cellulose binding protein and can be used to separate cellulose from a sample.
In another embodiment, the ligand may be covalently attached to the oil bodies. For example, the ligand may be a small organic molecule such as biotin. Biotinylated oil bodies can be used to separate avidin from a sample.
The present invention also includes modified oil bodies for use as an affinity matrix. Accordingly, the present invention includes a composition comprising oil bodies associated with a molecule, such as a ligand molecule or a target molecule. In one embodiment, the composition comprises oil bodies covalently attached to a ligand molecule, such as biotin.
The present invention also includes an affinity matrix for use in separating a target molecule from a sample, comprising oil bodies that can associate with the target molecule. The affinity matrix may additionally include a ligand molecule associated with the oil bodies, wherein the ligand molecule is capable of associating with the target molecule.
Other objects, features and advantages of the present invention will become apparent from the following detailed description and attached drawings. It should be understood, however, that the detailed description and associated examples are given by way of illustration only, and various changes and modifications thereto falling within the scope of the invention will become apparent to those skilled in the art. In addition, reference is made herein to various publications, patents and patent applications which are hereby incorporated by reference in their entirety.